Understanding the mechanisms that underlie abnormalities of electrical conduction in the heart is the key to the development of effective antiarrhythmic therapies. During the last decade, significant progress has been made in imaging electrical excitation waves in the heart using voltage-sensitive fluorescent dyes. However, until recently imaging using voltage-sensitive dyes was limited primarily to the epicardial surface. The goal of the proposed study is to develop a technology that would enable optical imaging of electrical excitation throughout the myocardial wall. Specifically, this technology should image the filaments, or organizing centers of vortex-like electrical activity. These are widely believed to be responsible for the initiation and maintenance of ventricular fibrillation, and the filaments are a key to their behavior. To address the technical challenges of this novel technology we propose a coordinated project involving the research groups of Dr. A. Pertsov (PI) from the Department of Pharmacology, SUNY Upstate Medical University, who pioneered the three-dimensional imaging of vortex-like excitation in chemical excitable systems and in the heart; Dr. D. Boas at the Harvard Medical School and the Massachusetts General Hospital NMR Center, an expert in optical tomography; Dr. L. Loew at the Center for Biomedical Imaging Technology, University of Connecticut Health Center (Co-PI), a leader in the development of voltage-sensitive probes and optical imaging; and the group of Dr. D. Weitz at the Department of Physics, Harvard University (Co-PI), renowned for their expertise in optical imaging and multiple-scattering media. The specific aims of the project are: 1) to create realistic computer models for reconstructing 2D optical images from 3D distributions of the transmembrane potential in myocardial tissue (forward problem), 2) to apply diffusive optical tomography to 3D reconstruction of the actual electrical activation in the heart (inverse problem); 3) to design, synthesize and test in myocardial tissues a family of near-infrared voltage-sensitive dyes optimized for 3D imaging of electrical activation in the heart; 4) to explore two-photon fluorescence and second-harmonic generation for 3D imaging of electrical activity in cardiac myocytes and tissues at subcellular and sub-millimeter scales. Successful completion of this project will break ground for a new technology, the 3D imaging of electrical activation in the heart.